If a neutrally charged biological cell is placed in a uniform electric field, such as provided by a pair of electrodes which are both planar, the biological cell does not move toward one electrode or another because the attractive forces from both electrodes are the same.
On the other hand, if a neutrally charged biological cell is placed in a non-uniform electric field, such as provided by two electrodes which are both not planar, as shown in PRIOR ART FIG. 1, the biological cell forms a dipole, is attracted to one electrode with greater attractive force than the other, and moves towards the electrode having the greater attractive force.
Such a use of a non-uniform electric field is used in dielectrophoresis, and the concept of using dielectrophoresis to align living cells, followed by a fusion/electroporation pulse, to fuse cells has been in the literature since early 1970's. This process is used to produce hybrids of two different cell types for therapeutic purposes, for hybridoma production for producing monoclonal antibodies, for nuclear fusion, and for producing other hybrid cells. Dielectrophoresis is the process of applying an electrical force on neutrally charged particles such as living cells. The force from dielectrophoresis results from applying a non-uniform electric field that separates charges inside the cells forming a dipole. After the dipole has been formed, the non-uniform electric field then moves the cells towards the highest or lowest electric field intensity. This movement is dependent on the relative conductivities and permittivities of the medium and the biological cells or particles. The dielectrophoretic force is a function of the electric field squared so electric field polarity is not important. The force is a function of the relative conductivities and permitivities of the medium and the particles or cells. The conductivities and permitivities are also a function of frequency of the applied electric field. Typically, an AC voltage wave, such as a sine wave, is applied across electrodes to produce this alternating electric field. The sine wave voltage, frequency, and duration are optimized for specific cell types. After the AC wave is applied to align the cells, one or more fusion/electroporation pulses are applied to form pathways in the cell membranes in which membranes from both cells commingle. These pathways permit the contents of the cells to mix forming a hybrid cell. Following the fusion pulses, another AC field can be applied to hold the cells together while the fused cells stabilize. In some cases, the AC voltage has been linearly increased or decreased to prevent damage to the cells due to a sudden application of a field.
Examples of cell fusion applications include hybridoma production and nuclear transfer. A recent application for electrofusion is to produce therapeutic hybrids for cancer immunotherapy. These hybrids are produced from cancer tumor cells and immune system dendritic cells in an ex vivo process. Each treatment requires a large number of viable hybrids, which results in a new requirement for high efficiency in the hybrid production process.
There are a number of techniques (electrical, mechanical, chemical) available to perform cell fusion. This invention relates to electrical means. The current electric art uses a voltage waveform generator connected to an electrode device. With respect to relevant known electrical, mechanical, and chemical techniques, the following U.S. Patents and published PCT application are of particular interest and are incorporated herein by reference:
4,326,934Apr. 27, 1982Pohl4,441,972Apr. 10, 1982Pohl4,764,473Aug. 16, 1988Matschke et al4,784,954Nov. 15, 1988Zimmermann5,304,486Apr. 19, 1994Chang6,010,613Jan. 4, 2000Walters et alWO 00/60065Oct. 12, 2000Walters et al
From the above, it is known to use pre-fusion electric field waveforms that have either a constant amplitude, see PRIOR ART FIG. 3, or a linearly increasing amplitude, see PRIOR ART FIG. 4. FIG. 5 illustrates an overall general PRIOR ART protocol for carrying out cell fusion using electric field waveforms, wherein a pre-fusion electric field waveform is followed by a fusion/electroporation pulse, which is followed by a post-fusion electric field waveform.
Nevertheless, efficiency of cell fusion following a constant amplitude or a linearly increasing amplitude of pre-fusion electric field waveforms cannot deliver the higher efficiencies required in such applications as therapeutic hybrid production for cancer immunotherapy. In this respect, it would be desirable if pre-fusion electric field waveforms were provided for biological cells which increases cell fusion efficiency over biological cells treated with a constant amplitude or a linearly increasing amplitude pre-fusion electric field waveform.
More specifically with respect to U.S. Pat. No. 5,304,486 of Chang, it is noted that FIG. 2E of Chang discloses a linear low voltage presine AC waveform, a high voltage linear electroporating AC waveform, and a low voltage linear post-poration AC waveform. The invention of Chang is confined solely to the fusion/electroporation pulses. Chang discloses only a linear, low voltage presine AC waveform. Chang does not disclose a non-linear low voltage presine AC waveform. Chang does not focus attention on the presine AC waveform, other than a nominal statement thereof.
The first process in any cell fusion system is to bring the cells into contact. In any case, sufficient force must be applied to each cell to overcome the negative surface charge. Merely applying a uniform electric field will not move a cell because the net charge of the cell is zero. Thus from the definition of electric field, there is no force applied:Force=(Electric Field)*(Charge)
However, a non-uniform field moves the positive ions inside each cell to one side and the negative ions to the opposite side producing a dipole, as shown in PRIOR ART FIG. 1. Once the dipole is induced, a net force is exerted, on the cell because the intensity of the field is greater on one side than the other. The movement of cells in one direction causes the cells to concentrate in an area. Since the cells are now dipoles, the negative side of one cell will attract the positive side of another cell overcoming the negative surface charge, as shown in PRIOR ART FIG. 2. The non-uniform electric field is produced by the electrode device. The non-uniformity is a function of the electrode configuration, as shown in PRIOR ART FIGS. 1 and 2.
Generally, the cell types to be fused are placed in a low conductive medium (less than 0.01 S/m) to minimize ohmic heating that may harm the cells and that causes turbulence thus reducing the number of fused hybrids. In this respect, it would be desirable for biological cells being subjected cell fusion to be treated so as to reduce heating during cell alignment and cell membrane contact.
The waveform generator has two functions. The first is to produce the AC voltage waveform that is converted into an AC field by the electrode device. This AC field then brings the cells into alignment/contact. The second function is to produce a pulse voltage that electroporates the cell membrane, fusing the cells. In some cases another AC voltage is produced after the fusing pulse to hold the cells in alignment until the fusion products become viable or stable.
One of the factors for successful fusion is the membrane contact between the adjacent cells. The closer this contact before the fusion pulse is applied, the higher the efficiency of fusion. In U. Zimmermann, et al., “Electric Field Induced Cell to Cell Fusion”, J. Membrane Biol. 67, 165-182 (1982), Zimmermann points out that increasing the AC wave electric field strength just before the fusion pulse may be the optimum approach. Clearly, it would be desirable for biological cells that are to undergo cell fusion to be pretreated with pre-fusion electric field waveforms which bring abort increased cell membrane contact without turbulence or heating.
In addition, there are a number of reasons why it is not desirable to immediately provide a high amplitude alignment waveform to cells that are to undergo cell fusion. A first reason is a mechanical reason. That is, immediate application of a high amplitude alignment waveform causes extreme force to be exerted on the cells, causing the cells to move rapidly towards an electrode. This rapid cell movement causes turbulence forces in the medium surrounding the cells. The turbulence forces do not allow complete pearl chains of cells to form, and the turbulence forces cause already formed pearl chains of cells to break up.
A second reason why it is not desirable to immediately provide a high amplitude alignment waveform to cells that are to undergo cell fusion is that such a high amplitude alignment waveform causes heating to occur in the media in which the biological cells are suspended. Heating also causes turbulence which does not permit complete pearl chains of aligned cells to form and causes already formed pearl chains to break up. The heat in the heated up media also reduces cell viability.
In view of the above, it would be desirable to avoid the mechanical forces, turbulence, and heating which result from immediately applying a high amplitude alignment waveform to biological cells that are to undergo cell fusion.
Thus, while the foregoing body of prior art indicates it to be well known to use pre-fusion electric field waveforms prior to carrying out cell fusion with en electroporation pulse, the prior art described above does not teach or suggest a dielectrophoresis waveform for cell fusion which has the following combination of desirable features: (1) provides pre-fusion electric field waveforms for biological cells which increase cell fusion efficiency over biological cells treated with a constant amplitude or a linearly increasing amplitude pre-fusion electric field waveforms; (2) avoids the mechanical forces, turbulence, and heating which result from immediately applying a high amplitude alignment waveform to biological cells that are to undergo cell fusion; (3) reduces heating of biological cells being treated with pre-fusion electric field waveforms for increasing cell alignment and cell membrane contact prior to being subjected to cell fusion; and (4) increase cell membrane contact between biological cells treated with pre-fusion electric field waveforms prior to undergoing cell fusion. The foregoing desired characteristics are provided by the unique non-linear dielectrophoresis waveform for cell fusion of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.
Additional U.S. patents that are of interest include:
4,561,961Dec. 31, 1985Hofmann5,001,056Mar. 19, 1991Snyder et al5,589,047Dec. 31, 1996Coster et al5,650,305Jul. 22, 1997Hui et al
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